CN115963255A - Preparation method and application of human basophilic granulocyte immunofluorescence sample - Google Patents
Preparation method and application of human basophilic granulocyte immunofluorescence sample Download PDFInfo
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Abstract
The invention belongs to the technical field of medical treatment, and discloses a preparation method and application of a human basophilic granulocyte immunofluorescence sample. The preparation method comprises the following steps: sorting peripheral blood basophilic granulocyte of human, culturing and collecting cell precipitate; adding paraformaldehyde to fix cells, and collecting cell precipitates; staining cell nuclei with DAPI; adding 0.75-1% of low-melting-point agar for resuspending cells to obtain cell suspension; and dripping the cell suspension on a glass slide for mounting and solidifying at room temperature for a moment to obtain a sample to be detected. The cell suspension device has extremely low requirement on the number of cells, can fix the suspended cells, can adjust the volume of heavy suspension according to the experiment requirement so as to adjust the cell density, is convenient to obtain the low-melting-point agarose, has low melting point, and can uniformly disperse the cells by heavy suspension; after the mounting, the gel is quickly formed at about 30 ℃, the suspended cells can be fixed and conveniently observed on the premise that the appearance of the suspended cells is not damaged, the cost is low, and the steps are simple and convenient.
Description
Technical Field
The invention relates to the technical field of medical treatment, in particular to a preparation method and application of a human basophil immunofluorescence sample.
Background
Basophils (Basophil, ba), a small population of peripheral blood leukocytes, are one of the rarest cell types, account for less than 1% of human leukocytes, and are the major effector cells of the immune response. Basophils can migrate to local tissues (such as lymph nodes, skin, etc.) to play a role, which plays an important role in allergic reactions. In recent years, the function of basophils in innate immunity, adaptive immunity, and in particular, in immunomodulation has attracted considerable attention, and the associated research efforts have led to significant advances and compelling efforts. Activation is a key step in the development of the biological function of basophils in different disease states. The mechanism of activation has also been studied, from classical IgE-mediated activation, to multi-pathway activation mechanisms, which include activation by a series of signals mediated by cytokines, antibodies, proteases, ligands for Toll-like receptors, and complement. In clinical and basic experimental studies in the prior art, the expression of CD203c and CD63 molecules is detected by a flow cytometer to indirectly mark the activation and degranulation states of basophils.
At present, the confocal laser scanning microscope has been widely applied to the fields of molecular cell biology such as immunology, morphology, physiology, and the like, but since the imaging is performed in a point-by-point, line-by-line, and plane-by-plane scanning manner, it is necessary to ensure that a sample is fixed in a plane during the imaging, so that the confocal laser scanning microscope cannot be applied to the imaging of suspended cells, especially rare cells (such as basophils) where the sample is precious. The suspension cell imaging technology mainly includes two methods: firstly, utilize cell smear centrifuge to make the suspension cell be close to and hug closely on the slide that polylysine was handled under the effect of centrifugal force, but the effect of centrifugal force can make cell morphology change, and can't make cell homodisperse in the plane to the influence is to the observation of cell structure. And secondly, adding the suspended cells into a culture dish coated by gelatin, and performing cell imaging after the suspended cells are settled to the bottom of the culture dish under the action of gravity. However, the method has poor stability, and a large amount of suspended cells still leave the bottom of the culture dish in the movable culture dish, so that the cells cannot be uniformly dispersed, and the observation effect is influenced.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method and application of a human basophil immunofluorescence sample, which can directly, objectively and accurately realize the visualization of the ingestion of exosomes after the activation of human basophils.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a method for preparing a human basophil immunofluorescence sample, comprising the following steps:
s1: sorting peripheral blood basophilic granulocyte, culturing and collecting cell precipitate;
s2: adding paraformaldehyde to fix cells, and collecting cell precipitates;
s3: staining cell nuclei with DAPI;
s4: adding 0.75-1% of low-melting-point agar for resuspending cells to obtain cell suspension;
s5: and (5) sealing the sheet to obtain the finished product.
Compared with the traditional sample preparation method, the preparation method disclosed by the invention has extremely low requirement on the number of cells, can fix the suspended cells, can adjust the resuspension volume according to the experimental requirement so as to adjust the cell density, and has a wider detection range. The low-melting-point agarose is convenient to obtain, has a low melting point, can maintain a liquid state at 37-72 ℃, and can uniformly disperse cells through heavy suspension; after the mounting, the gel is formed at about 30 ℃, so that the suspended cells can be fixed and are convenient to observe on the premise that the appearance of the suspended cells is not damaged; steps S2-S4 can be carried out in a 1.5ml EP tube, and the whole preparation process does not need smear, spin-drying, permeation and other processes. The method has the advantages of low cost and simple and convenient steps, and is suitable for preparing the suspension cell sample with extremely small cell quantity.
As a preferred embodiment of the preparation method of the human basophil immunofluorescence sample, in the S1, basophils are selected from anticoagulated peripheral blood of normal people by a negative selection method, activated by Anti-IgE stimulation, added with a PKH 26-labeled exosome, cultured, washed and collected with cell precipitates.
Preferably, the concentration of the Anti-IgE is 0.8-1.2 mu g/mL.
As a preferred embodiment of the preparation method of the human basophil immunofluorescence sample, 4% paraformaldehyde is added into S2, the mixture is fixed at room temperature for 10min to 15min, and cell precipitates are collected by centrifugation.
As a preferred embodiment of the preparation method of the human basophilic granulocyte immunofluorescence sample, the DAPI staining solution is added into the S3, the cells are resuspended, the cells are incubated at normal temperature in a dark place for 5-10 min, the supernatant is discarded by centrifugation, and the cell precipitate is cleaned and collected.
As a preferred embodiment of the method for preparing a human basophil immunofluorescence sample, the DAPI staining solution is prepared by mixing a mixture of a DAPI staining solution and a fluorescent reagent, wherein the volume ratio of the DAPI staining solution to the fluorescent reagent is 4-6: 1 PBS buffer solution and DAPI staining solution.
In a preferred embodiment of the method for preparing a human basophil immunofluorescence sample according to the present invention, in S4, the method for preparing the low melting point agarose comprises: adding the low-melting-point agarose powder into ultrapure water, fully dissolving and uniformly mixing to obtain the agarose gel; the temperature of the low-melting-point agarose is 37-72 ℃; the dosage of the low-melting-point agarose is 5-7 mu L.
In a preferred embodiment of the method for preparing a human basophil immunofluorescence sample according to the present invention, in S5, the method for mounting the patch comprises: heating the glass slide and the cover glass to 37-72 ℃, dripping the cell suspension obtained in the step S4 on the glass slide, covering the glass slide, and standing in a dark place at room temperature until the low-melting-point agarose is solidified to obtain the agarose gel.
In a second aspect, the invention provides a human basophil immunofluorescence sample prepared by the preparation method.
In a third aspect, the invention provides the use of the preparation method and the human basophil immunofluorescence sample in the microscopic observation of exosome uptake after the activation of human basophils.
As a preferred embodiment of the application of the present invention, the observation is performed by a confocal laser microscope.
Compared with the prior art, the invention has the beneficial effects that:
compared with the traditional sample preparation method, the preparation method disclosed by the invention has extremely low requirement on the number of cells, can fix the suspended cells, can adjust the resuspension volume according to the experimental requirement so as to adjust the cell density, and has a wider detection range. The low-melting-point agarose is convenient to obtain, has low melting point, can maintain liquid state at 37-72 ℃, and can uniformly disperse cells by resuspension; after the mounting, the gel is formed at about 30 ℃, so that the suspended cells can be fixed and are convenient to observe on the premise that the appearance of the suspended cells is not damaged; steps S2-S4 can be carried out in a 1.5ml EP tube, and the whole preparation process does not need smear, spin-drying, permeation and other processes. The method has the advantages of low cost and simple and convenient steps, and is suitable for preparing the suspension cell sample with extremely small cell quantity.
Drawings
FIG. 1 is an immunofluorescence electron micrograph of the PKH 26-labeled exosomes taken up after activation of human basophils obtained by the method of the present invention;
in the figure, blue is (the nucleus of) a DAPI dye-labeled basophil; the red color is PKH 26-tagged exosomes.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It should be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are commercially available; low melting point agarose was purchased from Invitrogen.
Example 1: preparation method of human basophilic granulocyte immunofluorescence sample
The method for preparing the human basophil to take up the PKH 26-labeled exosome after being activated comprises the following specific steps:
(1) Sorting, culturing and collecting human peripheral blood basophilic granulocyte
Selecting basophilic granulocyte from anticoagulated peripheral blood of normal human by negative selection method, activating with Anti-IgE stimulation of 1 μ g/mL, adding PKH 26-labeled exosome, placing at 37 deg.C, and 5% CO 2 After culturing for 3-24 hours, collecting cells by using a 1.5mL EP tube, centrifuging at room temperature for 300g multiplied by 10min, discarding supernatant, adding 1mL PBS buffer solution for washing, completely sucking the supernatant after centrifuging, and taking the precipitate for the next step;
(2) Fixing the device
Adding 1ml of 4% paraformaldehyde for fixation, fixing at room temperature for 10min, centrifuging at 300gx 10min, discarding the supernatant, adding 1ml of PBS buffer solution for washing, centrifuging, completely sucking the supernatant, and taking the precipitate for the next step;
(3) DAPI staining of nuclei
PBS buffer: DAPI stain =5:1, preparation. Adding 100 mu L of prepared DAPI dye solution, gently blowing and beating to resuspend the precipitated cells, incubating at normal temperature in dark for 5-10min, centrifuging at 300gx 10min, discarding the supernatant, adding 1ml of PBS buffer solution for washing, completely sucking the supernatant after centrifugation, and taking the precipitate for the next step;
(4) Resuspension of a suspension
1% Low melting agarose preparation: 1g of commercial low-melting-point agarose powder is added into 100ml of ultrapure water to be fully dissolved and uniformly mixed, and the mixture is subpackaged and frozen in a refrigerator at the temperature of 20 ℃ below zero. When in use, the mixture is placed on a metal bath at 72 ℃ for dissolution, and the liquid state is maintained at 37 ℃. Placing the cell sediment with the supernatant completely sucked in a metal bath at 37 ℃, adding 5-7 mu L of 1% low-melting-point agarose, and gently resuspending the cell sediment to avoid bubbles;
(5) Sealing sheet
Placing the glass slide and the cover slip on a metal bath at 37 ℃, dropwise adding the cell suspension on the glass slide, covering the glass slide, transferring to a room temperature, keeping in the dark for 1-2 min, and obtaining a sample to be detected after 1% of low-melting-point agarose is solidified.
(6) Confocal laser microscopy
Olympus confocal laser microscope FV3000, scanning resolution is 1024X 1024, multiplying power is 60X, DAPI fluorescence is excited at 405nm, PKH26 fluorescence is excited at 551nm, working voltage is adjusted, and picture results are recorded and saved.
Example 2: preparation method of human basophilic granulocyte immunofluorescence sample
The method for preparing the human basophil to take up the PKH 26-labeled exosome after being activated comprises the following specific steps:
(1) Sorting, culturing and collecting human peripheral blood basophilic granulocyte
Selecting basophilic granulocyte from anticoagulant peripheral blood of normal human by negative selection method, activating by Anti-IgE stimulation, adding PKH 26-labeled exosome, placing at 37 deg.C, and removing CO 5% 2 After culturing for 12 hours, collecting cells by using a 1.5mL EP tube, centrifuging at room temperature for 300g multiplied by 10min, discarding supernatant, adding 1mL PBS buffer solution for washing, completely sucking the supernatant after centrifuging, and taking the precipitate for the next step;
(2) Fixing the device
Adding 1ml of 4% paraformaldehyde for fixation, fixing at room temperature for 10min, centrifuging at 300gx 10min, discarding the supernatant, adding 1ml of PBS buffer solution for washing, centrifuging, completely sucking the supernatant, and taking the precipitate for the next step;
(3) DAPI staining of nuclei
PBS buffer: DAPI stain =5:1, preparing. Adding 100 mu L of prepared DAPI dye solution, gently blowing and beating to resuspend the precipitated cells, incubating at normal temperature for 10min in dark place, centrifuging for 300gx 10min, discarding the supernatant, adding 1ml of PBS buffer solution for washing, centrifuging, completely sucking the supernatant, and taking the precipitate for the next step;
(4) Resuspension
1% Low melting agarose preparation: 1g of commercial low-melting-point agarose powder is added into 100ml of ultrapure water to be fully dissolved and uniformly mixed, and the mixture is subpackaged and frozen in a refrigerator at the temperature of 20 ℃ below zero. When in use, the mixture is placed on a metal bath at 72 ℃ for dissolution, and the liquid state is maintained at 37 ℃. The cell sediment with the supernatant sucked off is placed in a metal bath at 37 ℃, 7 mu L of 1 percent low-melting-point agarose is added to lightly resuspend the cell sediment, and bubbles are avoided;
(5) Sealing sheet
Placing the glass slide and the cover glass on a metal bath at 37 ℃, dropwise adding the cell suspension on the glass slide, covering the glass slide, transferring to a room temperature, keeping in the dark for 2min, and obtaining a sample to be detected after 1% of low-melting-point agarose is solidified.
(6) Confocal laser microscopy
An Olympus laser confocal microscope FV3000 with a scanning resolution of 1024 x 1024 and a multiplying power of 60 x, DAPI fluorescence is excited at 405nm, PKH26 fluorescence is excited at 551nm, working voltage is adjusted, and picture results are recorded and stored.
FIG. 1 is a schematic representation of immunofluorescence obtained using the method of this example, of human basophils activated and then taken up in PKH 26-labeled exosomes, in which the blue color is (the nucleus of) the DAPI dye-labeled basophils; the red color is PKH 26-tagged exosomes. As can be seen from the pictures, the cell morphology is intact, and it can be clearly seen that human basophils take up PKH 26-labeled exosomes, which are mainly located in the basophil cytoplasm. And the group B is an immunofluorescence electron microscope image obtained after diluting the cell suspension of the group A by one time and mounting, and the group C is an immunofluorescence electron microscope image obtained after diluting the cell suspension of the group B by one time and mounting. Cells with different dilution ratios are uniformly dispersed in a visual field, and observation effects of different dilution ratios can be obtained according to different resuspension volumes. After mounting, 1% low-melting point agarose is condensed into gel, so that basophilic granulocytes are fixed in a plane and cannot be separated from an observation plane, and the basophilic granulocytes are not floated because of the movement of the observation process.
Example 3: preparation method of human basophilic granulocyte immunofluorescence sample
The difference from example 1 is that in step (4), 5. Mu.L of 0.75% low melting agarose was added to gently resuspend the cell pellet.
Compared with the traditional sample preparation method, the preparation method of the human basophilic granulocyte immunofluorescence sample has extremely low requirement on the number of cells, can fix the suspended cells, and can adjust the resuspension volume and further adjust the cell density according to the experiment requirement. The 0.75 to 1 percent of low-melting point agarose is convenient to obtain materials, has low melting point, can maintain liquid state at 37 ℃, and can uniformly disperse cells by heavy suspension; the gel is formed at about 30 ℃ after the sealing, the suspension cells can be fixed and conveniently observed on the premise that the appearance of the suspension cells is not damaged, the cost is low, the steps are simple and convenient, and the method is suitable for preparing suspension cell samples with extremely small cell quantity.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A preparation method of a human basophil immunofluorescence sample is characterized by comprising the following steps:
s1: sorting peripheral blood basophilic granulocyte of human, culturing and collecting cell precipitate;
s2: adding paraformaldehyde to fix cells, and collecting cell precipitates;
s3: staining cell nuclei with DAPI;
s4: adding 0.75-1% of low-melting-point agar for resuspending cells to obtain cell suspension;
s5: and sealing the sheets to obtain the finished product.
2. The method for preparing human basophil immunofluorescence sample according to claim 1, wherein in S1, the basophil is selected from anticoagulated peripheral blood of normal human by negative selection method, activated by Anti-IgE stimulation, added with PKH 26-labeled exosome, cultured, washed and collected.
3. The method for preparing a human basophil immunofluorescence sample according to claim 1, wherein 4% paraformaldehyde is added to S2, and the sample is fixed at room temperature for 10min to 15min, and cell precipitation is collected by centrifugation.
4. The method for preparing the human basophil immunofluorescence sample according to claim 1, wherein the DAPI staining solution is added into the S3, the cells are resuspended, the cells are incubated for 5min to 10min at normal temperature in a dark place, the supernatant is discarded by centrifugation, and the cell precipitate is washed and collected.
5. The method for preparing the human basophil immunofluorescence sample according to claim 4, wherein the DAPI staining solution is prepared by mixing the following components in a volume ratio of 4-6: 1 PBS buffer solution and DAPI staining solution.
6. The method for preparing human basophil immunofluorescence sample according to claim 1, wherein in the S4, the low melting point agarose is prepared by: adding the low-melting-point agarose powder into ultrapure water, fully dissolving and uniformly mixing to obtain the agarose gel; the temperature of the low-melting-point agarose is 37-72 ℃.
7. The method for preparing a human basophil immunofluorescence sample according to claim 1, wherein in the step S5, the mounting method comprises: heating the glass slide and the cover glass to 37-72 ℃, dripping the cell suspension obtained by the S4 on the glass slide, covering the glass slide, and standing in a dark place at room temperature until the low-melting-point agarose is solidified, thus obtaining the agarose gel.
8. The human basophil immunofluorescence sample prepared by the preparation method of any one of claims 1 to 7.
9. The method according to any one of claims 1 to 7, or the use of the human basophil immunofluorescence sample according to claim 8 for the microscopic uptake of exosomes following activation of human basophils.
10. Use according to claim 9, wherein the observation is performed using a confocal laser microscope.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113310963A (en) * | 2021-05-31 | 2021-08-27 | 四川大学华西医院 | Improved immunofluorescence detection method for neutrophil NETs |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113310963A (en) * | 2021-05-31 | 2021-08-27 | 四川大学华西医院 | Improved immunofluorescence detection method for neutrophil NETs |
| CN113310963B (en) * | 2021-05-31 | 2023-12-01 | 四川大学华西医院 | Improved neutrophil NETs immunofluorescence detection method |
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